The main long-term goals of this research are (1) to develop a better understanding of the control mechanisms operating during infection of E. coli by bacteriophage T4 and (2) to isolate and characterize nucleases in this system which appear potentially useful for specific fragmentation of DNA and for sequence studies. For the last three years, I have worked toward identifying and further purifying the nucleases which initiate the degradation of cytosine-containing DNA after T4 infection of E. coli. These nucleases are relatively specific for sites found about every 10 to the 6th power daltons in a variety of DNA's. I hope to further characterize the nature of these sites and determine whether or not they are between genes by isolating the fragments produced when the nucleases attack fd and 0X 174 single-stranded and replicating-form DNA, annealing them to intact strands of the opposite polarity bearing various amber mutations, and measuring growth in non-amber - permissive host, thus mapping the fragments genetically. I then plan to look at the action of these enzymes on such other DNA's as T7, SV40, and Xenopus mitochondrial DNA. It appears possible that these enzymes could be used to help isolate various specific genes. Other aspects of the program include: - (1) I have shown that mutants lacking these nucleases synthesize intact C-containing T4-DNA in vivo, but make almost no late proteins, as determined by slab-gel electrophoresis, and grossly overproduce gene 32 protein. I am using this system to study some of the control mechanisms affecting phage protein synthesis in vivo and in vitro. (2) Using mutants unable to degrade the host DNA to try and determine some of the factors which so efficiently turn off all host functions in T4-infected cells. (3) Determining whether or not any of the new E. coli endonucleases I have found during the course of this work have useful specificities and, if so, further purifying and characterizing them.